Cardiovascular and pulmonary diseases can disrupt the supply of O2 to tissues. Inadequate O2 supply can threaten cell survival and trigger organ failure. All mammalian cells can detect decreases in oxygen availability (hypoxia), and activate adaptive responses that protect them from the consequences of O2 deprivation. However, the underlying mechanisms of hypoxia sensing are not known. We hypothesize that mitochondria function as hypoxia sensors in the cell. These organelles appear to trigger adaptive responses by initiating a signaling cascade involving an increased release of reactive oxygen species (ROS) from the electron transport chain. We propose to study the molecular mechanisms underlying this response, and the role of the mitochondrial hypoxia sensor in the regulation of adaptive responses to hypoxia. Aim 1 will test whether conformational changes in mitochondrial Complex III induced by hypoxia cause an increase in ROS release to the intermembrane space and the cytosol. These oxidants may trigger adaptive responses including the stabilization of Hypoxia-Inducible Factor-1 alpha (HIF-1a) and the activation of hypoxic pulmonary vasoconstriction. This aim will be tested by protein crystallization studies of intact Complex III under different O2 concentrations. We will study the requirement for Complex III in hypoxia sensing by employing a new mouse model with conditional deletion of RISP, a functional component of the Complex required for ROS generation. Aim 2 will test whether increased hypoxia-induced ROS release from Complex III leads to an increase in oxidant signaling in the mitochondrial intermembrane space and the cytosol. This will be tested using novel protein-based redox sensors targeted to subcellular compartments. Aim 3 will clarify the signaling pathways linking the increase in ROS signaling during hypoxia and the downstream stabilization of HIF-1a. We hypothesize that Phospholipid Hydroperoxide Glutathione Peroxidase (PHGPx) functions as a signal transduction messenger in the ROS-HIF-1a pathway, transmitting the hypoxia-induced ROS to the stabilization of HIF-1a. We will also test the hypothesis that oxidation of HIF-1a itself, through the redox modification of a cysteine thiol, contributes to the regulation of its stability. Excessive or inadequate activation of the hypoxia sensing pathway contributes to cardiovascular and pulmonary disease pathogenesis, justifying these studies in terms of clinical relevance. PUBLIC HEALTH RELEVANCE: The cells in the body use oxygen to generate energy. When diseases of the lungs, blood vessels or the heart interfere with the supply of oxygen to tissues, the cells of the affected tissues can fail to perform normally, or even die. Each cell in the body has the ability to sense how much oxygen it receives. When the supply of oxygen to a cell decreases, a sensor in the cell activates a set of adaptive responses that protect it from damage in the event that oxygen becomes critically limiting. In this regard, the cellular oxygen sensor is important for normal health. However, in some diseases excessive or insufficient activation of the oxygen sensor can contribute to the effects of the disease. The mechanism of cellular oxygen sensing is not understood. This project seeks to test the hypothesis that mitochondria, organelles in the cell that normally consume oxygen, are the site of oxygen sensing. We have developed a new mouse model to test this hypothesis, which allows us to inactivate the ability of mitochondria to generate oxygen free radicals. We hypothesize that free radicals generated by the mitochondria are responsible for activating the adaptive responses to low oxygen conditions. In particular, we will test the hypothesis that a protein termed HIF, which is responsible for activating protective genes in response to low oxygen, is controlled by the free radical signaling from the mitochondria. Collectively, these studies will provide new information regarding the mechanisms of oxygen sensing in cells, which is important in normal tissues and in disease states.